Attenuation of West Nile Virus NS2B/NS3 Protease by Amino Terminal Copper and Nickel Binding (ATCUN) Peptides

West
Nile virus NS2B/NS3 protease (WNVP) is a viable target for
the development of antiviral compounds. To that end, catalytic metallopeptides
that incorporate the copper-binding ATCUN motif into either the N-
or C-terminus of known WNVP targeting peptides have been developed
as new families of peptide-based inhibitors. Each metallopeptide was
evaluated based on its inhibitory constant (KI), time-dependent inactivation of the protein, Michaelis–Menten
parameters, and the ability to oxidatively modify WNVP. Following
catalytic inactivation of WNVP, sequencing by LC-MS/MS demonstrated
active site residues Ser135, Thr134, and Thr132, as well as residues
in the S2 binding pocket, to be modified by oxidative chemistry. Results
from a DNPH-based assay to detect oxidative damage showed the formation
of carbonyls in WNVP treated with metallopeptides. These results suggest
that the metallopeptides are attenuating WNVP activity by irreversible
oxidation of amino acids essential to substrate binding and catalysis.